VISUALIZATION OF HIV-1 INTEGRATION IN REAL-TIME PROJECT SUMMARY / ABSTRACT Retroviral infections, including HIV and HTLV, continue to be a pandemic problem. While several drug therapies are able to treat HIV-1 infection, the virus's propensity to develop resistance mutations remains challenging. A clear priority for the HIV-1 treatment arsenal is to identifying novel drug targets. Stable integration of a retroviral donor cDNA into the host chromosome is absolutely required for a productive infection. Nearly three decades of research have established that retroviral integration is extremely inefficient in vivo and in vitro. Integration is catalyzed by the retrovirus encoded integrase (IN), which in many retroviruses forms a tetramer complex with the two viral cDNA long terminal repeat (LTR) ends (termed an intasome). The IN protein removes two 3' nucleotides and catalyzes end joining (strand transfer) of the resulting recessed 3' hydroxyls across one major groove of the target DNA separated by 4-6 bp. HIV-1 integration requires a host protein co-factor PSIP1/LEDGF/p75 for stable intasome assembly and to target integration to chromatin marked with the histone H3(K36) trimethylation post-translational modification (PTM). Remarkably, the prototype foamy virus (PFV) remains the only complete intasome structure. While anti- retroviral drugs that target the HIV-1 enzyme integrase also inhibit the PFV integrase, it remains unclear whether the integration mechanics of these respective retroviral intasomes are similar. We have used novel single molecule analytical tools to demonstrate that the PFV intasome catalyzes the two strand transfer events during integration in quick succession. We also visualized PFV intasomes on a linear target DNA and determined that the vast majority of IN-mediated search events were nonproductive. Together these observations suggested that target site selection limits PFV integration. We propose to extend our single molecule analysis to the biophysical mechanism of HIV-1 integration. Our preliminary studies have already identified significant differences between PFV and HIV-1, suggesting that the biophysical analysis of HIV-1 is likely to be more relevant to human health. Several important questions will be addressed in this new grant application: 1.) What are the protein dynamics of HIV-1 IN during the DNA target search and integration? 2.) What are the viral DNA dynamics during the DNA target search and integration process? 3.) What constitutes an efficient DNA target site? 4.) What factors influence the DNA target search process? 5.) What are the dynamics of intasome targeting on chromatin? We propose to utilize several innovative single molecule imaging systems to visualize HIV-1 integration in real- time. We will examine DNA lesions and mechanically altered DNA structures that may mimic the preferred target DNA configuration as well as chromatin containing histones with specific PTMs. These studies are designed to fully interrogate the animated processes associated with HIV-1 integration.